AU2007327367B2

AU2007327367B2 - Immunization protocol against the 4 dengue serotypes

Info

Publication number: AU2007327367B2
Application number: AU2007327367A
Authority: AU
Inventors: Veronique Barban; Remi Forrat; Bruno Guy; Jean Lang
Original assignee: Sanofi Pasteur Inc
Current assignee: Sanofi Pasteur Inc
Priority date: 2006-12-01
Filing date: 2007-11-30
Publication date: 2013-05-09
Anticipated expiration: 2027-11-30
Also published as: MY180698A; CN101541344A; ZA200902540B; IL198466A0; BRPI0718927A2; US20080131460A1; JP2010511022A; KR20090087890A; AU2007327367A1; MX2009004223A; FR2909286A1; IL230052A0; CA2668570A1; JP5269803B2; TW200829599A; EP2099483A1; US7718359B2; IL198466A; AR064009A1; EP2099483B1

Abstract

The invention relates to a method to induce a protection against the 4 dengue serotypes in a patient, comprising: (a) the administration of a monovalent vaccine comprising a vaccine virus of a first dengue serotype, and (b) the administration of a tetravalent vaccine comprising vaccine viruses of the four serotypes of dengue, wherein the administration (b) is implemented at least 30 days to at most 12 months after the first administration (a).

Description

W02008/065315 PCT/FR2007/052431 Immunization protocol against the 4 dengue serotypes Method of immunization against the 4 serotypes of dengue fever The invention relates to a method for 5 inducing protection against the 4 serotypes of dengue fever in a patient, comprising: (a) a first administration of a monovalent vaccine comprising a vaccinal virus of a first serotype of dengue fever, 10 (b) a second administration of a tetravalent vaccine comprising vaccinal viruses of the four serotypes of dengue fever, and in which the second administration (b) is made between at least 30 days and not more than 12 months 15 after the first administration (a). Dengue fevers are caused by four viruses of the flavivirus genus which are of similar serological type but differ from the antigen point of view (Gabler et al., 1988, in: Epidemiology of arthropod-borne viral 20 disease. Monath TPM, editor, Boca Raton (FL) : CRC Press: 223-60; Kautner et al., 1997, J. of Pediatrics, 131: 516-524; Rigau-P6rez et al., 1998, Lancet, 352: 971-977; Vaughn et al., 1997, J. Infect. Dis., 176: 322-30) . Infection with a serotype of dengue fever may 25 produce a spectrum of clinical disease from non-specific viral syndrome to severe fatal hemorrhagic disease. The incubation period for dengue fever after a mosquito bite is approximately 4 days (from 3 to 14 days) . Dengue fever is characterized by a two-phase 30 fever, headaches, pains in various parts of the body, prostration, eruptions and lymphadenopathy (Kautner et al., 1997, J. of Pediatrics, 131: 516-524; Rigau-P~rez et al., 1998, Lancet, 352: 971-977). The viremic period is of the same as the febrile period (Vaughn et al., 35 1997, J. Infect. Dis., 176: 322-30) . Cure of dengue fever is complete after 7 to 10 days, but prolonged asthenia is normal. Reduced leukocyte and platelet numbers frequently occur.

W02008/065315 PCT/FR2007/052431 -2 Hemorrhagic dengue fever is a severe febrile disease characterized by homeostasis abnormalities and an increase in vascular permeability which can lead to hypovolemia and hypotension (dengue fever with shock 5 syndrome), often complicated by severe internal bleeding. The mortality rate for hemorrhagic dengue fever can reach 10% without treatment, but is 5 1% in most centers with experience of treatment (WHO Technical Guide, 1986. Dengue haemorrhagic fever: 10 diagnosis, treatment and control, p. 1-2. World Health Organization, Geneva, Switzerland). Routine laboratory diagnosis of dengue fever is based on isolation of the virus and/or the detection of antibodies specific to dengue fever virus. 15 Dengue is the second most important infectious tropical disease after malaria, more than half of the world's population living in areas where there is a risk of epidemic transmission. There are estimated to be 50-100 million cases of dengue fever every year, 20 500,000 patients hospitalized for hemorrhagic dengue fever, and 25,000 deaths. Dengue fever is endemic in Asia, the Pacific, Africa, Latin America and the Caribbean. Dengue fever virus infections are endemic in more than 100 tropical countries and hemorrhagic dengue 25 fever has been documented in 60 of these countries (Gubler, 2002, TRENDS in Microbiology, 10: 100-103; Monath, 1994, Proc. Natl. Acad. Sci. , 91: 2395-2400) . A number of well-described factors would appear to be implicated in dengue fever - population growth, 30 unplanned and uncontrolled urbanization, in particular associated with poverty, an increase in air travel, lack of effective mosquito control and deterioration of sanitary and public health infrastructure (Gubler, 2002, TRENDS in Microbiology, 10: 100-103) . Travellers 35 and expatriates are increasingly being warned about dengue fever (Shirtcliffe et al., 1998, J. Roy. Coll. Phys. Lond., 32: 235-237). Dengue fever has been one of the main causes of febrile diseases among American W02008/065315 PCT/FR2007/052431 -3 troops during deployments in tropical areas where dengue fever is endemic (DeFraites et al., 1994, MMWR, 1994, 43: 845-848). The viruses are maintained within a cycle 5 involving humans and Aedes aegypti, a domestic mosquito which bites during the daytime, and prefers to feed on man. Infection in man is initiated by injection of the virus during the blood meal of an infected Aedes aegypti mosquito. The salivary virus is mainly 10 deposited in the extravascular tissues. The first category of cells to be infected after inoculation are the dentritic cells, which then migrate to the lymphatic ganglia (Wu et al., 2000, Nature Med., 7: 816-820). After initial replication in the skin and 15 lymphatic ganglia, the virus appears in the blood in the course of the acute febrile stage, generally for 3 to 5 days. Along with the dentritic cells, monocytes and macrophages are among the first targets of dengue fever 20 virus. Protection against homotypic reinfection is complete and probably lasts a lifetime, but cross-protection between the different types of dengue lasts from less than a few weeks to a few months (Sabin, 1952, Am. J. Trop. Med. Hyg., 1: 30-50). As a 25 consequence, an individual may become infected with a different serotype. A second infection due to dengue fever is in theory a risk factor for the development of severe dengue fever. However, hemorrhagic dengue fever is multifactorial - factors include the strain of virus 30 involved and the age, immune status and genetic predisposition of the patient. Two factors play a major role in the occurrence of hemorrhagic dengue fever rapid viral replication with a high level of viremia (the severity of the disease being associated with the 35 level of viremia; Vaughn et al., 2000, J. Inf. Dis., 181: 2-9) and a major inflammatory response with the release of high levels of inflammatory mediators (Rothman and Ennis, 1999, Virology, 257: 1-6) . There is WO2008/065315 PCT/FR2007/052431 -4 no specific treatment against dengue fever. Treatment for dengue fever is symptomatic, with bed rest, control of the fever and pain through antipyretics and analgesics, and adequate drinking. The treatment of 5 hemorrhagic dengue fever requires balancing of liquid losses, replacement of coagulation factors and the infusion of heparin. Preventive measures are currently based on control of the vector and personal protection measures which 10 are difficult to apply and are costly. No vaccine against dengue fever has at present been approved. Given that the four serotypes of dengue fever are in circulation in the world and that they have been reported as being involved in cases of hemorrhagic 15 dengue fever, vaccination should ideally confer protection against the four serotypes of dengue fever virus. When immunizing with a tetravalent vaccine, it may happen that the response is induced predominantly 20 against only one or at most 3 serotypes. There is therefore a need for a method which makes it possible to reduce interference between the different serotypes and makes it possible to induce neutralizing antibodies against the 4 serotypes of dengue fever. 25 The inventors have found that it is possible to generate an immune response comprising antibodies neutralizing the 4 serotypes when the vaccinal formulation which is intended to induce a response against the 4 serotypes is administered after 30 preliminary immunization with an attenuated living vaccine of only one serotype, the second immunization being made 30 days to 12 months after the first administration. The inventors have in particular shown that 35 tetravalent DEN-1,2,3,4 immunization after monovalent DEN-2 immunization induces responses against the four serotypes in all the monkeys immunized. Conversely, tetravalent immunization alone only induced a W02008/065315 PCT/FR2007/052431 -5 satisfactory response against two out of 4 serotypes, even after a booster. The immune response generated by the method according to the invention is therefore both 5 quantitatively and qualitatively greater (covers all serotypes). In accordance with a first object, this invention therefore relates to a method making it possible to induce a neutralizing antibody response against the 4 10 serotypes of dengue fever in a patient, comprising: (a) a first administration of a monovalent vaccine comprising a vaccinal virus of a first serotype of dengue fever, (b) a second administration of a tetravalent 15 vaccine comprising vaccinal viruses of the 4 serotypes of dengue fever, and in which the second administration (b) is made at least 30 days and not more than 12 months after the first administration (a). 20 According to a particular embodiment of the method of immunization according to the invention, the said vaccinal virus used in the first administration (a) is selected from the group comprising vaccinal viruses of dengue fever of serotype 1 or 2. 25 According to another particular embodiment of the method of immunization according to the invention, the said vaccinal virus used in the first administration (a) is selected from the group comprising strains VDV1 and VDV2. 30 According to another particular embodiment of the method according to the invention, the said vaccinal viruses used in the tetravalent vaccine are selected from the group comprising ChimerivaxTM DEN-1,2,3 and 4. According to another particular embodiment of the 35 method according to the invention the quantity of vaccinal viruses of dengue fever of serotypes 1, 2, 3 and 4 lies within a range from 103 to 106 CCIDso. According to another particular embodiment of the W02008/065315 PCT/FR2007/052431 -6 method according to the invention, the monovalent vaccine comprises 104 CCID 50 of VDV1 or VDV2 and the tetravalent vaccine comprises 105 CCIDso of ChimerivaxTM DEN-1,2,3 and 103 CCIDso of ChimerivaxTM DEN-4. 5 According to another embodiment of the method according to the invention, the second administration (b) is made 30 to 60 days after the first administration (a). Another object of the present invention is an 10 immunization kit against dengue fever virus comprising a box containing at least (a) a first container holding a monovalent composition or vaccine comprising a vaccinal virus of a first serotype of dengue fever, (b) a second container holding a tetravalent composition or 15 vaccine comprising vaccinal viruses for the 4 serotypes of dengue fever. According to one embodiment, the kit according to the invention comprises at least: (a) a first container holding a monovalent 20 vaccine comprising a VDV1 or VDV2 vaccinal virus, (b) a second container holding a tetravalent vaccine comprising the 4 ChimerivaxTM DEN-1,2,3 and 4. According to a particular embodiment, the kit according to the invention comprises a monovalent 25 vaccine comprising 104 CCID 5 of VDV1 or VDV2 and a tetravalent vaccine comprising 105 CCID5o of ChimerivaXTM DEN-1,2,3 and 10 3

CCID

5 o of ChimerivaxTM DEN-4. This invention therefore also relates to use of dengue fever vaccinal viruses for the manufacture of a 30 monovalent vaccine and a tetravalent vaccine for immunization against dengue fever virus in which the monovalent vaccine comprises a vaccinal virus of a first serotype of dengue fever, the tetravalent vaccine comprises vaccinal viruses of the 4 serotypes of dengue 35 fever and in which the tetravalent vaccine is administered at least 30 days and not more than 12 months after administration of the monovalent vaccine. The invention will now be described in more detail W02008/065315 PCT/FR2007/052431 -7 in the description which follows. Definitions "Dengue fever viruses" or "DEN" are positive 5 single-strand RNA viruses belonging to the Flavivirus genus of the family of flaviviridae. The genome in RNA contains a type I end member at the 5' extremity but has no poly-A tail at the 3' extremity. The organization of the genome comprises the following 10 elements: non-coding region (NCR) 5', structural proteins (capsid (C), pre-membrane/membrane (prM/M), envelope (E)) and non-structural proteins (NSl-NS2A NS2B-NS3-NS4A-NS4B-NS5) and NCR 3'. The viral genome RNA is associated with the capsid proteins to form a 15 nucleocapsid. As in the case of flaviviruses, the DEN viral genome codes an uninterrupted coding region which is translated into a single polyprotein. In the context of this invention, by "vaccinal dengue fever virus" is meant any viral form of dengue 20 fever virus which is capable of inducing a specific immune response comprising neutralizing antibodies, preferably all viral forms of dengue fever virus which can be used in the context of an immunization program in man against infection by a dengue fever virus. By 25 vaccinal dengue fever viruses are therefore meant inactivated viruses, attenuating viruses or recombinant proteins such as the envelope protein of dengue fever virus. A vaccinal virus is regarded as being 30 "inactivated" if it no longer replicates in permissive cells. A vaccinal virus is regarded as being "attenuated" if after growth at 37 0 C or 39 0 C in Huh-7, VERO and/or C6/36 liver cells the said vaccinal virus has a maximum 35 titer which is at least 10 times less than maximum titer obtained with the wild-type parent strain under the same culture conditions and as measured using the same method for determining titer. A vaccinal virus W02008/065315 PCT/FR2007/052431 -8 which has diminished growth in at least one of the three cell types identified above is therefore regarded as being "attenuated" in the context of this invention. A vaccinal virus which can be used in man has a 5 positive benefit/risk ratio, the said ratio generally satisfying statutory requirements for obtaining a marketing authorization. A vaccinal dengue fever virus used in the context of this invention is preferably a virus which has been attenuated in such a way that it 10 does not induce the disease in man. Advantageously, the said vaccinal virus only results in side effects of at most moderate intensity (i.e. medium to slight, or zero) in the majority of vaccinated individuals, while retaining its ability to induce a neutralizing antibody 15 response. Dengue fever vaccinal viruses which can be used in the context of this invention may be cited by way of non-restrictive examples: inactivated vaccinal viruses, attenuated vaccinal viruses such as the attenuated 20 strains VDV-1, VDV-2, the strains described for example in applications W002/66621, W00057904, W00057908, W00057909, W00057910, W002/0950075 and W002/102828, or chimeras. Chimeric viruses have the special feature that they have the characteristics of attenuated 25 viruses as defined above. All chimeric viruses expressing the envelope protein of a dengue fever virus and inducing an immune response comprising antibodies neutralizing the serotype from which the envelope protein originates may therefore be used in the context 30 of this invention. Mention may be made by way of non-restricting examples of: the dengue fever ChimerivaxTM such as described for example in patent application WO 98/37911, dengue/dengue fever chimeras such as described for example in patent applications 35 W09640933 and W00160847. The vaccinal virus of serotype 1 dengue fever may for example be the vaccinal strain VDV1 or a ChimerivaxTM DEN-1, in particular a YF17D/DEN-1 virus, or again a DEN-1 16007/PDK13 strain.

WO2008/065315 PCT/FR2007/052431 -9 The vaccinal virus for serotype 2 of dengue fever may for example be the vaccinal strain VDV2 or a ChimerivaxTM DEN-2, in particular a YF17D/DEN-2 virus, or again a DEN-2 16681/PDK53 strain. The vaccinal virus 5 of serotype 3 of dengue fever may be a ChimerivaxTM DEN-3, in particular a YF17D/DEN-3 virus. The vaccinal virus of serotype 4 of dengue fever may be a ChimerivaxTM DEN-4, in particular a YF17D/DEN-4 virus. Reference may be made to the applications identified 10 here for precise description of the strains mentioned and the processes for obtaining them. "VDV" or "Vero dengue vaccine" denotes an attenuated live dengue fever viral strain adapted to Vero cells (i.e. it is capable of replicating 15 reproducibly at a significant level on Vero cells) and capable of inducing a specific humoral response, including the induction of neutralizing antibodies, in primates and particularly in man. "VDV-1" is a strain obtained from a wild-type 20 DEN-1 16007 strain which has undergone 11 passes through PDK cells (DEN-1 16007/PDK11) and which has subsequently been amplified in Vero cells at 32 0 C, the RNA of which has been purified and transfected in Vero cells. The VDV-1 strain has 14 additional mutations in 25 comparison with the DEN-1 16007/PDK13 vaccinal strain (13 passes through PDK - Primary Dog Kidney - cells). The DEN-1 16007/PDK13 strain, also called "LAV1", has been described in patent application EP1159968 in the name of Mahidol University and has been filed with the 30 National Microorganisms Cultures Collection (CNCM) under number 1-2480. The complete sequence of the VDV-1 strain is given in sequence SEQ ID NO:1. This strain can easily be reproduced from that sequence. A process for preparing and characterizing the VDV-1 strain has 35 been described in the international patent application filed under number W02006/134433 in the names of Sanofi-Pasteur and the Center for Disease Control and Prevention.

W02008/065315 PCT/FR2007/052431 - 10 "VDV-2" is a strain obtained from a wild-type strain DEN-2 16681 which has undergone 50 passes through PDK cells (DEN-2 16681/PDK50), has been plate purified, and the RNA of which has been extracted and 5 purified, before transfection of said strain into Vero cells. The VDV-2 strain has subsequently been obtained by plate purification and amplification in Vero cells. The VDV-2 strain has 10 additional mutations in comparison with the DEN-2 16681/PDK53 vaccinal strain 10 (53 passes through PDK cells) , including 4 silent mutations. The DEN-2 16681/PDK53 strain, also known as "LAV2", has been described in patent application EP1159968 in the name of Mahidol University and has been filed with the National Microorganisms Cultures 15 Collection (CNCM) under number 1-2481. The complete sequence of the VDV-2 strain is given in sequence SEQ ID NO:2. The VDV-2 strain can easily be reproduced from that sequence. A process for preparing and characterizing the VDV-2 strain has been described in 20 the international patent application filed under number W02006/134433 in the names of Sanofi-Pasteur and the Center for Disease Control and Prevention. The VDV 1 and 2 strains are prepared by amplification in Vero cells. The viruses produced are 25 harvested and clarified from cell debris by filtration. The DNA is digested by treatment with enzymes. Impurities are eliminated by ultrafiltration. Infectious titers may be increased by a concentration method. After adding a stabilizer, the strains are 30 stored in lyophilized or frozen form before use and then reconstituted when needed. By "ChimeriVaX T M dengue" or "CYD" is meant a chimeric yellow fever (YF) virus which comprises the skeleton of a YF virus in which the sequences coding 35 for the pre-membrane and envelope proteins have been replaced by those of a DEN virus. Thus, a chimeric YF virus containing the prM and E sequences of a serotype 1 dengue fever strain (DEN-1) is called "CYD-1 or CYD W02008/065315 PCT/FR2007/052431 - 11 DEN1" . A chimeric YF containing the prM and E sequences of a DEN-2 strain is referred to as "CYD-2 or CYD DEN2" . A chimeric YF virus containing the prM and E sequences of a DEN-3 strain is referred to as "CYD-3 or 5 CYD DEN3" . A chimeric YF virus containing the prM and E sequences of a DEN-4 strain is referred to as "CYD-4 or CYD DEN4". The preparation of these dengue ChimeriVaxTM has been described in -detail in international patent applications WO 98/37911 and WO 03/101397, to which 10 reference may be made for a precise description of the processes for their preparation. The chimeras described in the examples have been generated by using prM and E sequences from strains DEN 1 PU0359 (TYP1140), DEN2 PUO218, DEN3 PaH881/88 and DEN 4 1228 (TVP 980) . Any 15 dengue fever virus strain may be used to construct chimeras in the context of this invention. Preferably, the chimeric YF virus comprises the skeleton of an attenuated yellow fever strain YF17D (Theiler M. and Smith H.H. (1937) J. Exp. Med., 65, 20 p. 767-786) (viruses YF17D/DEN-1, YF17D/DEN-2, YF17D/DEN-3, YF17D/DEN-4) . Examples of YF17D strains which may be used include YF17D204 (YF-Vax*, Sanofi-Pasteur, Swifwater, PA, USA; Stamaril*, Sanofi-Pasteur, Marcy l'Etoile, France; ARILVAXTM, 25 Chiron, Speke, Liverpool, UK; FLAVIMUNO, Berna Biotech, Bern, Switzerland; YF17D-204 France (X15067, X15062); YF17D-204,234 US (Rice et al., 1985, Science, 229: 726-733) , or again the related strains YF17DD (Genbank access number U17066), YF17D-213 (Genbank access number 30 U17067) and the strains YF17DD described by Galler et al. (1998, Vaccines, 16 (9/10) : 1024-1028) . Any other attenuated yellow fever virus strain which may be used in man may be used to construct chimeras in the context of this invention. 35 According to a particular embodiment, for each serotype used in the various administrations the vaccinal viruses are present in the vaccine in a quantity from 103 to 10s CCID 5 o - W02008/065315 PCT/FR2007/052431 - 12 According to a particular embodiment, vaccinal viruses VDV1 or VDV2 are present in the monovalent vaccine at a level of 104 CCID 5 o. According to a particular embodiment, ChimerivaxTM 5 DEN-1, 2, 3 are present in the tetravalent vaccine at a level of 10s CCIDso and ChimerivaxTM DEN-4 is present in the tetravalent vaccine at a level of 10 3 CCIDso . Each monovalent ChimeriVaxTM dengue fever vaccinal virus (serotypes 1, 2, 3 and 4) has been prepared by 10 amplifying each serotype in Vero cells. More specifically, the four viruses are produced separately in adhering Vero cells in a serum-free medium. The viral harvest, clarified from cell debris by filtration, is then concentrated and purified by 15 ultrafiltration and chromatography to remove the DNA from the host cells. After adding a stabilizing agent, the vaccinal strains are stored in a frozen or lyophilized form before use and then reconstituted as needed. The same process is applied to the four 20 chimeras. A dose, composition or vaccine is "monovalent" when in addition to a pharmaceutically acceptable excipient it contains a vaccinal virus of a single dengue fever serotype. A dose, composition or vaccine 25 is "tetravalent" when it contains vaccinal viruses of the four serotypes of dengue fever. Multivalent compositions are obtained by simple mixing of monovalent compositions. By "patient" is meant a person (child or adult) 30 who is likely to be infected by dengue fever, in particular a person at risk of infection, such as for example a person travelling in regions where dengue fever is present, or an inhabitant of those regions. The term therefore includes persons who are naive for 35 dengue fever virus and those who are not naive. Tetravalent immunization following initial monovalent. immunization W02008/065315 PCT/FR2007/052431 - 13 In a first aspect, this invention therefore relates to a method of immunization against dengue fever virus. The inventors have in fact shown in particular 5 that the administration of 4 serotypes 30 days to 12 months after the first administration of a monovalent vaccine makes it possible to obtain effective protection against the 4 serotypes. The method according to this invention is therefore of very 10 particular interest in the context of an immunization strategy against dengue fever. According to this invention, the first immunization may be performed using a monovalent composition or vaccine comprising a vaccinal virus of 15 any of the 4 serotypes of dengue fever, the second administration being performed with all 4 vaccinal serotypes. According to a particular embodiment, a serotype 1 or 2 dengue fever vaccinal virus, preferably serotype 2, is used for the first administration. 20 Preferably, the dengue fever vaccinal virus used in the first administration is an attenuated dengue fever virus and is not made up of a chimeric virus. According to a particular embodiment, strain VDV1 or VDV2, preferably strain VDV2, is used as the vaccinal virus 25 in the first administration. Attenuated living vaccinal viruses are used in the second administration, preferably chimeric viruses expressing antigens for the four serotypes of dengue fever virus, in particular ChimerivaxTm DEN1, 2, 3 and 30 4. According to particular embodiments, this invention therefore covers the following systems: - (a) VDV1 (b) CYD DEN-1, 2, 3 and 4 - (b) VDV2 (b) CYD DEN-1, 2, 3 and 4. 35 In the context of this invention, by "vaccinal composition" is meant a composition comprising an "immunoeffective quantity" of dengue fever vaccinal virus, that is to say a sufficient quantity of dengue W02008/065315 PCT/FR2007/052431 - 14 fever vaccinal virus to induce a specific immune response comprising neutralizing antibodies, which may be revealed for example by the seroneutralization test as described in Example 1 below. A serum is regarded as 5 being positive for the presence of neutralizing antibodies when the titer of neutralizing antibodies so determined is not less than 1:10 (unity: 1/dilution). The quantities of vaccinal strain are commonly expressed in terms of viral plaque forming units (PFU) 10 or doses infecting 50% of the tissue culture or again doses infecting 50% of the cell culture (CCID 5 0 ) . For example, compositions according to the invention may contain 10 to 106 CCID 5 o, in particular 103 to 105 CCID 5 o of dengue fever vaccinal virus of serotypes 1, 2, 3 or 15 4 for a monovalent or tetravalent composition. Thus, in the compositions or utilizations according to the invention the doses of dengue vaccinal viruses of serotypes 1, 2, 3 and 4 preferably each lie within a range from 10 to 106 CCID 5 o, such as 10, 102, 10 3, 104, 20 105 or 106 CCID 5 o, in particular within a range from 103 to 105 CCID 5 o. Vaccinal virus may be used at the same or different doses, which can be adjusted in relation to the nature of the vaccinal virus used and the intensity of the immune response obtained. 25 According to a particular embodiment of a method according to this invention, the quantities of attenuated live vaccinal virus in monovalent and tetravalent compositions or vaccines are 103 to 105

CCID

5 o. According to a particular embodiment, the 30 monovalent vaccine comprises 104 CCID 5 o of VDV1 or VDV2, preferably VDV2. According to a particular embodiment, the tetravalent vaccine comprises 105 CCID 5 o of ChimerivaxTM DEN-1, 2, 3 and 4. According to one advantageous embodiment, the tetravalent vaccine 35 comprises 105 CCID 5 O of ChimerivaxTM DEN-1, 2 and 3 and 103 CCID 50 of ChimerivaxM DEN-4. In the context of this invention, the second administration (b) is performed 30 days and not more W02008/065315 PCT/FR2007/052431 - 15 than 12 months after administration (a) . According to an advantageous embodiment, the second administration is performed 30 days to 60 days after the first administration (a). 5 The neutralizing antibody response is advantageously durable, that is to say it can be detected in serum up to at least 6 months after the second administration. Vaccinal viruses are administered in the form of 10 compositions or vaccines which can be prepared by any method known to those skilled in the art. Habitually, viruses, generally in lyophilized form, are mixed with a pharmaceutically acceptable excipient such as water or a phosphate-buffered saline solution, wetting agents 15 or stabilizing agents. By "pharmaceutically acceptable excipient" is meant any solvent, dispersing medium, charge, etc., which does not produce any secondary reaction, for example an allergic reaction, in humans or animals. The excipient is selected on the basis of 20 the pharmaceutical form chosen, the method and the route of administration. Appropriate excipients, and requirements in relation to pharmaceutical formulation, are described in "Remington: The Science & Practice of Pharmacy", which represents a reference work in the 25 field. Preferably, vaccinal compositions are prepared in injectable form, and may take the form of liquid solutions, suspensions or emulsions. The compositions may in particular comprise an aqueous solution buffered 30 in such a way as to maintain a pH between approximately 6 and 9 (as determined using a pH meter at ambient temperature). Although it is not necessary to add an adjuvant, the compositions may nevertheless include such a 35 compound, that is to say a substance which increases, stimulates or reinforces the cell or humoral immune response induced by the vaccinal virus administered simultaneously. Those skilled in the art will be able W02008/065315 PCT/FR2007/052431 - 16 to select an adjuvant which might be appropriate in the context of this invention from the adjuvants conventionally used in the field of vaccines. The compositions or vaccines according to the 5 invention may be administered by any means conventionally used in vaccination, for example parenterally (in particular intradermally, subcutaneously or intramuscularly), advantageously subcutaneously. Preferably, the compositions or 10 vaccines are injectable compositions administered subcutaneously, advantageously in the region of the left deltoid or right deltoid. The volume of vaccine composition administered will depend on the method of administration. In the 15 case of subcutaneous injections, the volume is generally between 0.1 and 1.0 ml, preferably approximately 0.5 ml. The optimum period for administering all serotypes 1 to 4 is approximately 1 to 3 months before exposure 20 to dengue fever virus. Vaccinations may be administered as a prophylactic treatment against infection by dengue fever virus in adults and children. Target populations therefore include persons who may be naive (i.e. not previously immunized) or non-naive with regard to 25 dengue fever virus. Booster administrations of dengue fever vaccinal viruses of serotypes 1 to 4 may also be used for example between 6 months and 10 years, for example 6 months, 1 year, 3 years, 5 years or 10 years after 30 administration of the second administration (b) according to the invention. Booster administrations will advantageously be performed using the same compositions or vaccines (i.e. the same vaccinal viruses) and preferably under the same conditions of 35 administration (anatomical sites and methods of administration) as used for the 2 "d administration (b). Interference phenomena may be explained by the dominance of one or more serotypes in relation to W02008/065315 PCT/FR2007/052431 - 17 others and are therefore independent of the technology used for preparation of the candidate vaccine (from VDV or ChimerivaxTM) . The method according to this invention can therefore be applied in general to all dengue fever 5 vaccinal viruses. This invention is therefore also intended to cover use of dengue fever vaccinal viruses for the manufacture of a monovalent vaccine and a tetravalent vaccine for immunization against dengue fever virus in 10 which the monovalent vaccine comprises the vaccinal virus of a first serotype of dengue fever, the tetravalent vaccine comprises vaccinal viruses for 4 serotypes of dengue fever, in which the tetravalent vaccine is administered at least 30 days and not later 15 than 12 months after administration of the monovalent vaccine. For a description of the vaccines and conditions of use in the context of use according to this invention, reference may be made to the description 20 provided in relation to the method of immunization according to the invention. According to another aspect, this invention has as its object an immunization kit against the four serotypes of dengue fever virus. The kit according to 25 this invention comprises compositions or vaccines as defined above in relation to the method of immunization proposed. The kit according to the invention therefore comprises a box containing various containers holding the compositions or vaccines and advantageously an 30 explanatory brochure including useful information for administration of the said compositions or vaccines. According to one embodiment, this invention therefore relates to a kit for immunization against dengue fever virus, a box containing at least (a) a 35 first container holding a monovalent vaccine comprising a vaccinal virus of a first serotype of dengue fever, and (b) a second container holding a tetravalent vaccine comprising vaccinal viruses for the 4 serotypes W02008/065315 PCT/FR2007/052431 - 18 of dengue fever. For a description of the vaccines, compositions or dengue fever vaccinal viruses which may be used in the kit according to the invention, reference may be made 5 to the description provided above in relation to the method of immunization according to the invention. According to a particular embodiment the kit according to the invention comprises at least: (a) a first container holding a monovalent 10 vaccine comprising a VDV1 or VDV2 vaccinal virus, and (b) a second container holding a tetravalent vaccine comprising the 4 ChimerivaxTM DEN-1, 2, 3 and 4. According to a particular embodiment, the kit according to the invention comprises at least one 15 monovalent vaccine comprising 104 CCID 50 of VDV1 or VDV2 and a tetravalent vaccine comprising 105 CCID 5 o of Chimerivax'M DEN-1, 2, 3 and 103 CCID 5 o of ChimerivaxTM DEN-4. The kits according to the invention may contain a 20 single example or several examples of the containers as described above. If the vaccines used are in lyophilized form, the kit will advantageously comprise at least one additional container holding the diluent which can be 25 used to reconstitute an injectable dose of vaccine. Any pharmaceutically acceptable diluent may be used for this purpose, conventionally water or a phosphate-buffered aqueous solution. The invention is illustrated by the following 30 example. Example 1: Immunization against the 4 serotypes of dengue fever virus by successive injection of a monovalent composition followed by a tetravalent 35 composition in monkeys Viremia and immunogenicity were tested in a monkey model. Viremia in particular has been identified as being one of the factors associated with the virulence W02008/065315 PCT/FR2007/052431 - 19 and severity of the disease in man, and therefore constitutes an important parameter which must be taken into consideration. As for immunogenicity, this is a key parameter in the context of evaluating the 5 protection imparted. 1.1 Materials and methods Experiments on monkeys were carried out in accordance with European Directives relating to animal 10 experiments. The immunizations were performed on cynomolgus monkeys (Macaca fascicularis) originating from Mauritania. The monkeys were placed in quarantine for six weeks prior to immunization. The monkeys were immunized subcutaneously with 15 0.5 ml of vaccine composition in the arm. After mild anesthesia with ketamine (Imalgene, Merial), blood was collected by puncture of the inguinal or saphenal veins. On days 0 and 28 following each immunization, 5 ml of blood were sampled in order to evaluate 20 antibody responses, while between days 2 and 10 1 ml of blood was sampled in order to evaluate viremia. The blood was collected on ice and preserved on ice until the serum was separated off. In order to do this, the blood was centrifuged for 20 minutes at 4 0 C and the 25 serum collected was stored at -800C until the time of the tests. Measurement of viremia Post-vaccination viremia was monitored by quantitative real time RT-PCT (qRT-PCR). Two sets of 30 initiators and sensors located in the NS5 gene of the DEN1 and DEN2 strains were used to quantify the RNA of VDV-1 and VDV-2 respectively. A third set of 2 initiators and 1 sensor located in the NS5 gene of the YF virus was used to quantify the RNA of CYD. Finally, 35 4 sets of initiators and specific sensors for the different CYD serotypes located at the junction of the E (DEN) / NS1 (YF) genes were used to identify the serotype in the samples positive for NS5 YF RNA (see W02008/065315 PCT/FR2007/052431 - 20 also Table 1). 7 plasmids containing the region targeted by each PCR, under the control of promoter T7, were transcribed in vitro to generate a series of synthetic RNA which were included in each RT-PCT test 5 as an internal reference. The synthetic RNA were determined by spectrophotometry, the quantity of RNA obtained was converted into the number of RNA copies and expressed as GEQ (genome equivalents). 0.140 ml of monkey serum were extracted using the 10 "Nucleospin 96 virusM" RNA extraction kit from Macherey Nagel according to the manufacturer's instructions, and then the purified RNA was eluted with 0.140 ml (0.090 ml, then 0.05 ml) of RNase-free water. In order to avoid repeated freeze/thaw cycles, a first 15 quantification was performed immediately after extraction on 5 p.l of the said RNA preparation. The remaining volume was frozen at 700C. In addition to the components of the "Qiagen QauntitectM probes" RT-PCR quantification kit (Qiagen), 20 the reaction mixtures contained 10 picomoles of each initiator, 4 picomoles of each sensor and 5 pl of RNA in a total volume of 25 p.l. In the case of the RNA under test, 5 p.l of the purified preparation were added directly to the reaction mixture without a prior 25 dilution stage. The synthetic RNAs were diluted 1/10 in RNAse-free water, and 7 dilutions containing approximately 10 to 106 GEQ in 5 pl were quantified in parallel in order to generate a calibration curve. The quantification reactions were carried out 30 using the ABIPrism 700 TM equipment from Applied Biosystem, using the following program: 500C/30 min, 950C/15 min, followed by 40 cycles of 950C/15 sec 600C/60 sec. The quantification limit for viral RNA in this 35 test is 2.9 to 3.3 logioGEQ/ml (800 to 2000 GEQ/ml; 4 to 10 GEQ/reaction), according to PCR targets (standard deviation: +/-0.3 logio). The correlation between infectious titer and the W02008/065315 PCT/FR2007/052431 - 21 quantification of viral RNA was established in parallel with the tests by analyzing 0.140 ml of samples of negative monkey serums (DO) to which a known quantity of infectious particles of the viruses used for 5 immunization (CYD or VDV) had been added. The said control serums were prepared in two dilutions containing approximately 1 PFU and approximately 100 PFU in 5 tl (2.3 and 4.3 logioPFU/ml, respectively) . In the tests used in the examples, the correlation 10 between GEQ and PFU is as follows: GEQ/PFU ratio 2.7 logo (i.e. 1 PFU = 500 GEQ) for sera positive for YF or CYDs; GEQ/PFU ratio 2.5 logio (i.e. 1 PFU = 320 GEQ) for sera positive for VDV1 or VDV2. The quantification limits are < 3.3 logioGEQ/ml 15 (i.e. < 4 PFU/ml) for YF and CYDs qRT-PCR, and < 2.9 logi 0 GEQ/ml (i.e. < 2.5 PFU/ml) for VDV1 and VDV2 qRT-PCR. The initiators and sensors used are shown in Table 1 below, in which the sense and anti-sense initiators 20 and the sensor are listed in order for each test.

W02008/065315 PCT/FR2007/052431 - 22 Table 1 sequence YF-NS5 sens 5' GCACOGATGTAACAGACTOAAGA (23 bases) YF NSS anti 5' CCAGGCCGAACCTGTCAT (18 bases) YF-NS5 5' Farn- COGACTGTGTGGTOOGOOOCATC -Tamra (22 bases) E CYD1- sens 5' CAT TOC AGT TGG CCT GOT AA (20 b) >- a) CYD1- antfi 5' CTT TOG CAA GAG AGA GOCT CAA OT (23 b) CYD1- 5' Farn-CCG ATC AAG GAT GCG CCA TCA-Tarnra (21 b) CYD2- sens 5' GTO GGA OTC GTG ACO CTG TA (20 b) C CYD2- anti 5' OTT GAT G0C GCA TCC TTG ATC (21 b) OCYD2 5' FarrTGG GAG TTA TGG TOG GCG CCG-Tamra (21 b) M CYD3- sens- 5' AAA ACA CTT CCA TOT CAT TTT CAT G (25b) C CYD3- anti: 5' GTT GAT 00 C GCA T CC TTG ATC (21 b) CYD3- 5'FarrTG CGATAGGAATTATCACACTCTATCTGGGAGC-Tamra (33b) CYD4- sens 5' CTT AGT ATT OTG GAT TOG CAC GAA (24 b) a, CYD4- anti: 5'GCG CCA ACT GTG AAA CCT AGA (21 b) CYD4- 5'-FarrAGAAAOAOCCCAATOOCAATGACGTGCAT-Tarnra (29 b) VDV1-NS5 sens 5' TCG CAA CAG CCT TAA CAG C (19 b) a DV1-NS5 anti 5'ACT ATC TCC CTC CCA TCC TTC ( 2 1 b) VDV1-NS5 5' Farr-TTC ACA CCA CTT CCA C-MGBINFQ (16 b) VDV2-NS5 sens 5' AAT GAC AGA CAC GAC TCC (18 b) o VDV2-NSS anti: 5' CCC AAA ACC TAC TAT CTT CAA C (22 b) VDV2-NS5 5' FarrTGG AAG TCG GCA CGT GA-MGBNFQ (17 b) Measurement of neutralizing antibodies 5 (seroneutralization test) (SN50) Conventionally, dengue fever antibodies are measured using the PRNT50 test (test of neutralization by reducing the number of PFU to 50%) . As this test is cumbersome and consumes much material, we have 10 developed the SN50 test based on a 50% reduction in the number of units measured in the CCID50 test. In a 96 well plate, 0.120 ml of each decomplemented serum is added to 0.480 ml of diluent (ISCOVE 4% SVF) in each well. Serial dilutions of a 15 factor 6 are performed by transferring 0.150 ml of serum into 0.450 ml of diluent. 450 pl of viral dilution containing 2.7 logo CCID50/ml are added to each well so as to obtain 25 CCID50/well. The plate is incubated at 370C for 1 hour. 0.1 ml of each dilution 20 is then distributed into 6 wells of a 96 well plate in which VERO cells have been seeded 3 days before the start of the experiment at a density of 8000 cells/well in 0.1 ml of ISCOVE 4% SVF medium. After 6 days W02008/065315 PCT/FR2007/052431 - 23 incubation at 37 0 C in the presence of 5% C0 2 , the cells are fixed using an ethanol/acetone (70/30) mixture at 4 0 C for 15 minutes, and then washed 3 times in PBS and incubated for 1 hour at 37 0 C in the presence of 0.05 ml 5 of a 1/2000 dilution of an anti-flavivirus monoclonal antibody (mAb 4G2 obtained from an ATCC H-B112 hybridoma). The plates are then washed twice and incubated for 1 hour at 37 0 C in the presence of 0.05 ml of a 1/1000 dilution of an anti-mouse IgG conjugated 10 with alkaline phosphatase. The lysis plaques are revealed by adding 0.05 ml of a stained substrate: BCIP/NBT. The neutralizing antibody titers are calculated using the Karber formula as defined below: 15 LogioSN50 = d + f/N (X + N/2) in which: d: represents the dilution providing 100% neutralization (that is 6 negative replicates, i.e. 20 presenting no signs of infection) f: represents the dilution factor as logo (e.g. dilution factor of 1:4, f = 0.6) N: represents the number of replicates/dilution (N = 6) 25 X: total number of wells having no sign of infection, with the exception of dilution d. The limit for viral detection is 10 SN50 (i.e. 1.0 logioSN50) . The viral strains used for neutralization were the 30 strains DEN1 16007, DEN2 16681, DEN3 16562 or DEN4 1036. In the case of the controls, the initial viral dilutions were re-titrated. The correlation between the neutralizing titer 35 measured in the SN50 test and the neutralizing titer measured conventionally in the PRNT50 test is: logioPRNT50 = logioSN50 + 0.2.

W02008/065315 PCT/FR2007/052431 - 24 1.2 Evaluation of simultaneous immunizations 2 groups of 4 monkeys of equivalent age and weight were immunized (see Table 2). Immunization was performed subcutaneously in the 5 arm using a 23G1 needle, with a quantity of 10s CCID 5 o for each CYD DEN 1 to 4 serotype for the tetravalent vaccine and a quantity of 104 CCIDso for the monovalent VDV-2. 10 Table 2: Composition of the groups and immunization protocol Monkeys Immunizations Groups DO D56 Tetravalent Monovalent Dengue Group 1 VDV 2 1234 ChimeriVax Tetravalent Tetravalent Dengue Dengue Group 2 1234 1234 ChimeriVax ChimeriVax The immunogenicity results obtained after one 15 immunization (DO+28) and two immunizations (D56+28) are shown in Table 3. The viremia results are provided in Table 4.

IT 0 Iq W 0D t- " m 0 m~ W z 0 0 0 - 1 w w w m Wl CN iLn Un 0 N CO O ZO 04) Im* ~ WL em c 14 mN00 04 to ULO C- o, co 0 CD o c 0 0 Ln to o oo 00 0 0 0 00 N WO 5, EO C104 0 0 -.- CC0 0) 0 ~ w- 0 a~ a) _ _E _2Ea <-<-E-<-- - - - - - -4 (Y) (D " N (lN IN (N N 1;4 e( i Cl) m m C C) Cl) CN 0 v v v v V v 0 C Hl Cl) (N (N( N ( (N) M. v v v v 00 (13o C14v 04LCol l C'. ) 0r CS! (N (N (N Co ) ') .) C'.) ('.) C') c'. oL v v v v v v v 0 v 0 c)v v v v v C C.- C.-CNN (C- ClJ C'.* C')m ( 0 ~ ~ ~ rOm-. ) Cl I C/) OD Vl l 0. _ I: i 0 -. W (Ni Cl

C

E C) v co ~ ~ O D Lq 1 6) q V V TO C: VD a VCD Vi V U)y 0D CCC >:r 0 cy C ( r 0 o 2 2Ez W02008/065315 PCT/FR2007/052431 - 27 In brief, the results can be summarized as follows: - The method of administration according to this invention brings about a qualitative and quantitative 5 increase in the neutralizing antibody response obtained with a system comprising two identical immunizations with tetravalent vaccine. - One CYD-1,2,3,4 immunization performed after an initial monovalent VDV2 immunization induces high level 10 responses against the four serotypes in all the monkeys, unlike a system comprising 2 immunizations of tetravalent vaccine. - As expected, the initial immunization performed with VDV2 induces a response which is almost 15 exclusively directed against serotype 2, with a low level of cross-reactivity against serotypes 1 and 4 in some animals. - Viremia is observed with VDV2 after initial immunization, and is predominantly caused by CYD-4 20 after the second administration (b) (group 1) . No noteworthy differences were observed in the viremia induced after a first immunization with tetravalent vaccine in naive animals (group 2). It can therefore be concluded that the system proposed by the invention 25 does not encourage the emergence of viremia of serotypes 1, 3 and 4 after the second administration. The examples therefore show that the method of immunization according to this invention improves the immunogenicity of the dengue fever vaccinal viruses 30 without adversely affecting the latter's safety. All the publications cited in the present application are incorporated in their entirety by way of reference.

. \Miil moven\NRPorbl\DCC\AM I5051134_1 doc.M4/2013 -27A Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or 5 steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general 10 knowledge in the field of endeavour to which this specification relates.

Claims

1. The use of dengue fever vaccinal viruses for the manufacture of a monovalent vaccine and a tetravalent vaccine for immunization against dengue fever virus, comprising: (a) a first administration of a monovalent vaccine comprising a vaccinal virus for a first serotype of dengue fever, wherein said vaccinal virus is selected from the group consisting of the strains VDVl and VDV2, (b) a second administration of a tetravalent vaccine comprising vaccinal viruses for the 4 serotypes of dengue fever, and in which the tetravalent vaccine is administered at least 30 days and not more than 12 months after administration of the monovalent vaccine.

2. The use as claimed in claim 1, in which the vaccinal viruses used in the tetravalent vaccine are CYD DEN-1, 2, 3 and 4.

3. The use as claimed in claim I or claim 2, in which the quantity of dengue fever vaccinal viruses of serotypes 1, 2, 3 and 4 lies within a range from 10 3 to 106 CCID 50 .

4. The use as claimed in any one of claims 1 to 3, in which the monovalent vaccine comprises 104 CCID 50 of VDVI or VDV2 and the tetravalent vaccine comprises 10 5 CCID 5 o of CYDDEN-1, 2, 3 and 4.

5. The use as claimed in any one of claims 1 to 3, in which the monovalent vaccine comprises 10 4 CCID 50 of VDV1 or VDV2 and the tetravalent vaccine comprises 105 CCID 50 of CYD DEN- 1, 2, 3 and 101 CCID 50 of CYD DEN-4.

6. The use as claimed in any one of claims 1 to 5, in which the second administration (b) is performed 30 to 60 days after the first administration (a).

7. An immunization kit against dengue fever virus comprising a box containing at least: H :AMT nerwoven\NRPonbl\D M \5511341 doc-8/4/20L3 -29 (a) a first container holding a monovalent vaccine comprising a vaccinal virus of a first serotype of dengue fever, wherein said vaccinal virus is selected from the group consisting of the strains VDVI and VDV2, (b) a second container holding a tetravalent vaccine comprising vaccinal viruses for the 4 serotypes of dengue fever, when used for sequential administration of the vaccines in (a) and (b), wherein the tetravalent vaccine of (b) is administered at least 30 days and not more than 12 months after administration of the monovalent vaccine of (a).

8. The immunization kit as claimed in claim 7, in which said second container holds a tetravalent vaccine comprising the 4CYD DEN-1,2,3 and 4.

9. The immunization kit as claimed in claim 8, in which the monovalent vaccine comprises 104 CCID 5 0 of VDVI or VDV2 and the tetravalent vaccine comprises 105 CCID 50 of CYD DEN-1,2,3 and 4.

10. The immunization kit as claimed in claim 8, in which the monovalent vaccine comprises 104 CCID 50 ofVDVI or VDV2 and the tetravalent vaccine comprises 105 CCID 5 o of CYD DEN-1,2,3 and 103 CCID 5 o of CYD DEN-4.

11. The immunization kit as claimed in any one of claims 7 to 10, wherein the tetravalent vaccine of (b) is administered 30 to 60 days after administration of the monovalent vaccine of (a).

12. A method for inducing a neutralising antibody response against the 4 serotypes of dengue fever in a patient, comprising: (a) a first administration of a monovalent vaccine comprising a vaccinal virus of a first serotype of dengue fever, wherein said vaccinal virus is selected from the group consisting of the strains VDV1 and VDV2, (b) a second administration of a tetravalent vaccine comprising vaccinal viruses of the 4 serotypes of dengue fever, and H :AM\mnterwoven\NRPorb\DCC\AM54)51134_l doc-M4/2013 - 30 in which the second administration (b) is made at least 30 days and not more than 12 months after the first administration (a).

13. The method as claimed in claim 12, in which the vaccinal viruses used in the tetravalent vaccine are CYD DEN-1, 2, 3 and 4.

14. The method as claimed in claim 12 or claim 13, in which the quantity vaccinal viruses of dengue fever of serotypes 1, 2, 3 and 4 lies within a range from 103 to 106 CCID 5 o.

15. The method as claimed in any one of claims 12 to 14, in which the monovalent vaccine comprises 10 4 CCID 5 o of VDV1 or VDV2 and the tetravalent vaccine comprises 10 5 CCID 5 o of CYD DEN-1, 2, 3 and 4.

16. The method as claimed in any one of claims 12 to 14, in which the monovalent vaccine comprises 104 CCID 50 of VDV1 or VDV2 and the tetravalent vaccine comprises 10' CCID 5 o of CYD DEN-1, 2, 3 and 103 CCID 50 of CYD DEN-4.

17. The method as claimed in any one of claims 12 to 16, in which the second administration (b) is performed 30 to 60 days after the first administration (a).

18. The use as claimed in claim 1, the immunization kit as claimed in claim 7 or the method as claimed in claim 12, substantially as herein described and with reference to any of the Examples and/or sequence listing.